Anti-cd33 antibodies and method for treatment of acute myeloid leukemia using the same

ABSTRACT

The present invention relates to antibodies that bind CD33. More particularly, the invention relates to anti-CD33 antibodies, fragments and homologues of these antibodies, humanized and resurfaced versions of these antibodies, functional equivalents and improved versions of these antibodies, immunoconjugates and compositions comprising these antibodies, and the uses of same in diagnostic, research and therapeutic applications. The invention also relates to a polynucleotide encoding these antibodies, vectors comprising the polynucleotides, host cells transformed with polynucleotides and methods of producing these antibodies.

This application is a Continuation Application of U.S. application Ser.No. 10/700,632, filed Nov. 5, 2003; which claims benefit of ProvisionalApplication No. 60/424,332 filed Nov. 7, 2002, the disclosure of each ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind CD33. Moreparticularly, the invention relates to anti-CD33 antibodies, fragmentsand homologues of said antibodies, humanized and resurfaced versions ofsaid antibodies, functional equivalents and improved versions of saidantibodies, immunoconjugates and compositions comprising saidantibodies, and the uses of same in diagnostic, research and therapeuticapplications.

In another aspect, the invention relates to a polynucleotide encodingthe antibodies, vectors comprising the polynucleotides, host cellstransformed with polynucleotides and methods of producing theantibodies.

BACKGROUND OF THE INVENTION

The leukocyte differentiation antigen CD33 is a 364 amino acidtransmembrane glycoprotein with sequence homology to members of thesialoadhesin family, including myelin-associated glycoprotein and CD22,as well as sialoadhesin itself (S. Peiper, 2002, Leucocyte Typing VII,White Cell Differentiation, Antigens, Proceedings of the SeventhInternational Workshop and Conference, Oxford University Press, p. 777).

Expression of CD33 appears to be highly specific to the hematopoieticcompartment, with strong expression by myeloid precursor cells (S.Peiper, 2002). It is expressed by myeloid progenitor cells such asCFU-GEMM, CFU-GM, CFU-G and BFU-E, monocytes/macrophages, granulocyteprecursors such as promyelocytes and myelocytes although with decreasedexpression upon maturation and differentiation, and mature granulocytesthough with a low level of expression (S. Peiper, 2002).

In contrast, pluripotent hematopoietic stem cells that give rise to“blast colonies” in vitro (Leary, A. G. et al., 1987, Blood 69:953) andthat induce hematopoietic long-term marrow cultures (Andrews R. G. etal., 1989, J. Exp. Med. 169:1721; Sutherland, H. J. et al., 1989, Blood74:1563) appear to lack expression of CD33.

While the specific function of CD33 is unknown, its homology tosialoadhesin suggested a role in carbohydrate binding characteristic ofthe lectin family, a role later confirmed (S. Peiper, 2002).

Importantly, anti-CD33 monoclonal antibodies have shown that CD33 isexpressed by clonogenic, acute myelogenous leukemia (AML) cells ingreater than 80% of human cases (LaRussa, V. F. et al., 1992, Exp.Hematol. 20:442-448).

Due to the selective expression of CD33, immunoconjugates that combinecytotoxic drugs with monoclonal antibodies that specifically recognizeand bind CD33 have been proposed for use in selective targeting of AMLcells. Such therapies are expected to leave stem cells and primitivehematopoietic progenitors unaffected. Immunoconjugates that utilizeanti-CD33 antibodies include anti-CD33-ricin immunoconjugates that havebeen shown to be highly lethal to AML cells (Roy, D. C. et al., 1991,Blood 77:2404; Lambert, J. M. et al., 1991, Biochemistry 30:3234), yetspare the stem cells that support normal hematopoiesis and hematopoieticreconstitution (LaRussa, V. F. et al., 1992, Exp. Hematol. 20:442-448).

Additional studies using immunoconjugates have shown rapid targeting ofradiolabeled anti-CD33 antibodies to leukemic blast cells in peripheralblood and marrow when administered i.v. (Scheinberg, D. A. et al., 1991,J. Clin. Oncol. 9: 478-490; Schwartz, M. A. et al., 1993, J. Clin.Oncol. 11:294-303). Rapid internalization of the antibody by the targetcell was also observed in in vitro studies (Tanimot, M. et al., 1989,Leukemia 3: 339-348; Divgi, C. R. et al., 1989, Cancer Res. Suppl. Vol.30: 404a). Evaluation of a humanized anti-CD33 antibody conjugated tothe potent antitumor antibiotic calicheamicin (Gemtuzumab ozogamicin) inpre-clinical studies demonstrated specific killing of leukemia cells inHL-60 cell cultures, HL-60 tumor xenografts in mice, and marrow samplesfrom AML patients (Hamann, P. R. et al., 2002, Bioconjugate Chem. 13:47-58).

Based on the positive results of these pre-clinical studies, Gemtuzumabozogamicin was evaluated in phase I and II clinical studies. In Phase Istudies, the major toxicity observed was myelosuppression due to theexpression of CD33 on myeloid progenitor cells (Sievers, E. L. et al.1999, Blood 93: 3678-3684; Sievers E. L. et al., 2001, J. Clin. Oncol.19: 3244-3254.). Phase II studies with a dose of 9 mg/m² i.v. over 4hours, repeated after 14 days, yielded a response rate of 30%. Marketingapproval of Gemtuzumab ozogamicin was granted by the FDA in May 2000with indication for the treatment of patients with CD33 positive AML infirst relapse who are 60 years of age or older and who are notconsidered candidates for cytotoxic chemotherapy. Post-marketing reportshave indicated the potential for significant toxicity, especiallyvenoocclusive disease (VOD), which has led to labeling revisions andinitiation of a patient surveillance program. Much of this toxicity maybe related to the drug component calicheamicin, which was shown to causehepatotoxicity in pre-clinical models, and therefore may not be a directresult of targeting CD33.

While the results discussed above suggest that immunoconjugatescomprising an anti-CD33 antibody and a cytotoxic drug may besuccessfully used in the treatment of AML, there is a need forimmunoconjugates that are both safe and effective. The present inventionis directed to these and other important ends.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideantibodies that specifically bind to CD33, and that may be used in thetreatment of AML.

Thus, in a first embodiment, there is provided an antibody, orepitope-binding fragment thereof, having the ability to bind CD33.

In a second embodiment, there is provided the murine antibody My9-6,which is fully characterized herein with respect to the amino acidsequences of both its light and heavy chain variable regions, the cDNAsequences of the genes for the light and heavy chain variable regions,the identification of its CDRs (complementarity-determining regions),the identification of its surface amino acids, and means for itsexpression in recombinant form.

In a third embodiment, there are provided humanized or resurfacedversions of the My9-6 antibody wherein surface-exposed residues of theMy9-6 antibody or epitope-binding fragments thereof are replaced in bothlight and heavy chains to more closely resemble known human antibodysurfaces. Such humanized antibodies may have increased utility, comparedto murine My9-6, as therapeutic or diagnostic agents. Humanized versionsof antibody My9-6 are also fully characterized herein with respect totheir respective amino acid sequences of both light and heavy chainvariable regions, the DNA sequences of the genes for the light and heavychain variable regions, the identification of the CDRs, theidentification of their surface amino acids, and disclosure of a meansfor their expression in recombinant form.

In a further embodiment, there are provided antibodies orepitope-binding fragments thereof comprising at least onecomplementarity-determining region having an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-6:

SYYIH, (SEQ ID NO: 1) VIYPGNDDISYNQKFXG, (SEQ ID NO: 2) wherein X is Kor Q EVRLRYFDV, (SEQ ID NO: 3) KSSQSVFFSSSQKNYLA, (SEQ ID NO: 4)WASTRES, (SEQ ID NO: 5) HQYLSSRT, (SEQ ID NO: 6)and having the ability to bind CD33.

In a further embodiment, there are provided antibodies orepitope-binding fragments thereof comprising at least one heavy chainvariable region and at least one light chain variable region, whereinsaid heavy chain variable region comprises threecomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOs:1-3, respectively,

SYYIH, (SEQ ID NO: 1) VIYPGNDDISYNQKFXG, (SEQ ID NO: 2) wherein X is Kor Q EVRLRYFDV, (SEQ ID NO: 3)and wherein said light chain variable region comprises threecomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOs:4-6, respectively,

KSSQSVFFSSSQKNYLA, (SEQ ID NO: 4) WASTRES, (SEQ ID NO: 5) HQYLSSRT, (SEQID NO: 6)

In a further embodiment, there are provided antibodies having a heavychain variable region that has an amino acid sequence that shares atleast 90% sequence identity with an amino acid sequence represented bySEQ ID NO:7:

QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFKGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREV RLRYFDVWGAGTTVTVSS,more preferably 95% sequence identity with SEQ ID NO:7, most preferably100% sequence identity with SEQ ID NO:7.

Similarly, there are provided antibodies having a light chain variableregion that has an amino acid sequence that shares at least 90% sequenceidentity with an amino acid sequence represented by SEQ ID NO:8:

NIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQYLSS RTFGGGTKLEIKR,more preferably 95% sequence identity with SEQ ID NO:8, most preferably100% sequence identity with SEQ ID NO:8.

In a further embodiment, antibodies are provided having a humanized orresurfaced heavy chain variable region that shares at least 90% sequenceidentity with an amino acid sequence represented by SEQ ID NO:9:

QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREV RLRYFDVWGQGTTVTVSS,more preferably 95% sequence identity with SEQ ID NO:9, most preferably100% sequence identity with SEQ ID NO:9.

Similarly, antibodies are provided having a humanized or resurfacedlight chain variable region that shares at least 90% sequence identitywith an amino acid sequence corresponding to SEQ ID NO:10:

EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLSS RTFGQGTKLEIKR,more preferably 95% sequence identity with SEQ ID NO:10, most preferably100% sequence identity with SEQ ID NO:10.

In a further embodiment, the present invention provides immunoconjugatescomprising a drug or prodrug covalently attached, directly or via acleavable or non-cleavable linker, to an antibody or epitope-bindingfragment thereof of the present invention. In preferred embodiments, thedrug or prodrug is a cytotoxic drug or prodrug such as a maytansinoid, ataxoid, CC-1065, a CC-1065 analog, dolastatin and a dolastatin analog.

In a further embodiment, the present invention provides a compositioncomprising an antibody or epitope-binding fragment thereof of thepresent invention and a drug or prodrug.

In a further embodiment, the present invention comprises pharmaceuticalcompositions comprising an antibody, epitope-binding fragment thereof orimmunoconjugate of the present invention, either alone or in combinationwith a drug or prodrug or other therapeutic agent, in the presence ofone or more pharmaceutically acceptable agent.

In a further embodiment, the present invention provides for an antibodyor epitope-binding fragment thereof that is labeled for use in researchor diagnostic applications. In preferred embodiments, the label is abiotin label, an enzyme label, a radio-label, a fluorophore, achromophore, an imaging agent or a metal ion.

In a further embodiment, the present invention provides methods forinhibiting the growth of a cell expressing CD33 through the use of anantibody, epitope-binding fragment thereof or immunoconjugate of thepresent invention, either alone or in combination with a drug or prodrugor other therapeutic agent, further alone or in the presence of one ormore pharmaceutically acceptable agent.

In an further embodiment, the invention provides methods for thetreatment of a subject having a disease wherein CD33 is expressedcomprising administering an antibody, an epitope-binding fragmentthereof or immunoconjugate of the present invention, either alone or incombination with another a drug or prodrug or another therapeutic agent,further alone or in the presence of one or more pharmaceuticallyacceptable agent. The disease may be one or more of, for example,myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML), or otherdisease yet to be determined in which CD33 is expressed.

The methods of treatment include in vivo, ex vivo and in vitroapplication of the antibodies, antibody fragments and immunoconjugatesof the present invention, either alone or in combination with a drug orprodrug or other therapeutic agent, further alone or in the presence ofone or more pharmaceutically acceptable agent.

In a further embodiment, a method of determining whether a biologicalsample contains a myelogenous cancer cell is provided wherein abiological sample is contacted with a diagnostic reagent, such as alabeled antibody or epitope-binding fragment thereof of the presentinvention, and the distribution of the reagent within the sample isdetected. This method may be used to diagnose a cancer such as acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).

In a further embodiment, antibodies or epitope-binding fragments thereofof the present invention are provided that have improved properties. Forexample, antibodies or epitope-binding fragments thereof having improvedaffinity for CD33 may be prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics.

Improved antibodies may also be prepared by affinity maturation of anantibody or epitope-binding fragment thereof of the present inventionthrough, for example, oligonucleotide-mediated site-directedmutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling anduse of mutator-strains of E. coli.

In a further embodiment, the present invention provides polynucleotidesencoding the antibodies or epitope-binding fragments thereof of thepresent invention, recombinant vectors comprising the polynucleotides,host cells transformed with the recombinant vectors and methods forproducing said antibodies and epitope-binding fragments thereof byculturing said host cells.

In a final embodiment, the present invention provides a method forobtaining CD33 from a biological material using an antibody orepitope-binding fragment thereof of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a competition binding experiment in whichbinding of ¹²⁵I-labeled My9-6 antibody (3×10⁻⁹ M) to CD33-positive U-937cells was assayed in the presence of increasing concentrations of eitherMy9 or My9-6 antibody.

FIG. 2 shows the My9-6 degenerate primers for the light chain signalsequence.

FIG. 3 shows the file names of the 127 antibody structures from theBrookhaven database that were used to predict the surface of the muMy9-6variable region.

FIG. 4 shows the PCR primers used to build the 16 resurfaced My9-6versions as well as chimeric My9-6 antibody.

FIG. 5 shows the plasmids used to build and express the humanizedantibodies. (A): the light chain cloning plasmid. (B): the heavy chaincloning plasmid. (C): the mammalian antibody expression plasmid.

FIG. 6A shows the Edman sequencing results compared with amino acidsequence derived from the RT-PCR generated cDNA clones for muMy9-6 lightchain.

FIG. 6B shows the results from the MS-MS sequence analysis of the 1319Da and 1122 Da peptide fragments containing CDR1 and CDR2 sequencesrespectively. CDR sequences are in bold.

FIG. 7 shows the results from the MS-MS sequence analysis of the 1788 Dapeptide and the corresponding sequence derived from two cDNA clones.

FIG. 8A shows the cDNA sequence and the deduced amino acid sequence (SEQID NO:95) of the light chain variable region for the murine My9-6antibody. The three CDRs are underlined.

FIG. 8B shows the cDNA sequence and the deduced amino acid sequence (SEQID NO:96) of the heavy chain variable region for the murine My9-6antibody. The three CDRs are underlined.

FIG. 9 shows the light and heavy chain CDRs as determined by the Kabatdefinitions.

FIG. 10 shows the light chain and heavy chain amino acid sequence forthe murine My9-6 antibody aligned with the germLine sequences for the8-27 and V102 genes. Dots (.) indicate sequence identity.

FIGS. 11A & B show the ten light chain (A) and heavy chain (B) antibodysequences most homologous to the muMy9-6 sequences that have solvedfiles in the Brookhaven database. Sequences are aligned in order of mostto least homology.

FIGS. 12A & B show the average accessibility for each Kabat position ofthe muMy9-6 antibody light (A) and heavy (B) chains. The relativesolvent accessibilities for each Kabat position of the ten mosthomologous light and heavy chain sequences were averaged and arepresented along the x axis.

FIG. 13A shows the residue solvent accessibilities for the ten mosthomologous light chain structures, calculated with the MC software, andshows the averages for each Kabat position, tabulated with Excel. Thistable presents the data for non-CDR positions with average solventaccessibilities greater than 25%. A surface residue is defined as aresidue with greater than a 30% average solvent accessibility. Positionswith 25%-35% average accessibilities were further analyzed bycalculating average accessibilities of structures only with identicalresidues at that position as well as in the two flanking positions oneither side. NA refers to identical flanking positions not available.Positions 15 and 70 required further calculations to arrive at the finalsurface predictions given in the last column.

FIG. 13B shows the residue solvent accessibilities for the ten mosthomologous heavy chain structures, calculated with the MC software, andshows the averages for each Kabat position, tabulated with Excel. Thistable presents the data for non-CDR positions with average solventaccessibilities greater than 25%. A surface residue is defined as aresidue with greater than a 30% average solvent accessibility. Positionswith 25%-35% average accessibilities were further analyzed bycalculating average accessibilities of structures only with identicalresidues at that position as well as in the two flanking positions oneither side. NA refers to identical flanking positions not available.

FIG. 14 shows the My9-6 framework surface residues that fall within 5 Åof a CDR residue.

FIG. 15 shows the top five human sequences extracted from the Kabatdatabase. Alignments were generated by SR (Pedersen, 1993). The muMy9-6residues that come within 5 Å of a CDR are underlined.

FIGS. 16A & B show the 16 humanized My9-6 light chain variable regionsequences (A) and the 16 humanized My9-6 heavy chain variable regionsequences (B) aligned with murine My9-6. The dots (.) represent sequenceidentity with humanized version 1.0. The surface residues that differ inmurine and human My9-6 are underlined.

FIG. 17 shows the My9-6 K_(D) values calculated by direct binding assayon HL-60 membranes and HL-60 whole cells, as well as competitive bindingassay on HL-60 membranes. N≧3 except for * where N=2.

FIG. 18 shows binding curves for huMy9-6 V1.0. (A): direct binding onHL-60 membranes. (B): direct binding on HL-60 whole cells. (C):competitive binding on HL-60 membranes.

FIG. 19 shows a comparison of binding of My9-6-DM1 with My9-6 antibodyon HL-60 cells.

FIG. 20 shows in vitro cytotoxicity of My9-6-DM1 toward CD33-expressinghuman tumor cells.

FIG. 21 shows the results of efficacy experiments of My9-6-DM1 in SCIDmice bearing HL-60 xenografts. The effect of My9-6-DM1(A) and unmodifiedMy9-6 antibody (C) on the growth of HL-60 tumors was evaluated. Mousebody weight was monitored as an indication of toxicity (B, D).

FIG. 22 shows a comparison of the efficacy of My9-6-DM1 with the freedrug maytansine in SCID mice bearing HL-60 xenografts (A). Mouse bodyweight was monitored as an indication of toxicity (B). Relapsed tumorsin two treated mice were treated with a second course of My9-6-DM1.

FIGS. 23 A & B show a comparison of anti-tumor efficacy of My9-6-DM1with standard chemotherapy in SCID mice bearing large HL-60 xenografts(A). Mouse body weight was monitored as an indication of toxicity (B).Relapsed tumors in two treated mice were retreated with a second courseof My9-6-DM1.

FIGS. 24A & B show anti-tumor efficacy of My9-6-DM1 compared withGentuzumab ozogamicin and standard chemotherapy in an HL-60 survivalmodel. HL-60 cells were intravenously injected into SCID mice. Indicatedtreatments were started 11 days following injection of cells. Treatmentswere i.v. daily×5 except for Gentuzumab ozogamicin (Q4D×3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel murine anti-CD33 antibody andhumanized versions of this antibody. Further provided are antibodiescomprising one or more of the CDRs of the murine anti-CD33 antibody orhumanized version thereof that specifically recognize and bind to CD33.

Murine My9-6 Antibody

The murine anti-CD33 antibody of the present invention, variouslydesignated herein as “My9-6”, “murine My9-6” and “muMy9-6”, is fullycharacterized with respect to the putative germline amino acid sequenceof both light and heavy chain variable regions (FIG. 10), amino acidsequences of both light and heavy chain variable regions (FIGS. 8A & B),the identification of the CDRs (FIG. 9), the identification of surfaceamino acids (FIGS. 13A & B), and means for its expression in recombinantform.

The My9-6 antibody has further been functionally characterized and shownto bind with high affinity to CD33 on the surface of CD33-positive U-937cells (FIG. 1). ¹²⁵-labeled My9-6 binds to U-937 cells and it iscompeted off the cells by unlabeled My9-6 and the previouslycharacterized anti-CD33 antibody My9 (BioGenex, cat. no. 267M).

The term “variable region” is used herein to describe certain portionsof antibody heavy chains and light chains that differ in sequence amongantibodies and that cooperate in the binding and specificity of eachparticular antibody for its antigen. Variability is not usually evenlydistributed throughout antibody variable regions. It is typicallyconcentrated within three segments of a variable region calledcomplementarity-determining regions (CDRs) or hypervariable regions,both in the light chain and the heavy chain variable regions. The morehighly conserved portions of the variable regions are called theframework regions. The variable regions of heavy and light chainscomprise four framework regions, largely adopting a beta-sheetconfiguration, with each framework region connected by the three CDRs,which form loops connecting the beta-sheet structure, and in some casesforming part of the beta-sheet structure. The CDRs in each chain areheld in close proximity by the framework regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen binding siteof antibodies (E. A. Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, 1991, NIH).

The “constant” region is not involved directly in binding an antibody toan antigen, but exhibits various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

Humanized My9-6 Antibody

Humanized versions of My9-6, variously designated herein as “huMy9-6”,and “humanized My9-6”, have also been prepared.

The goal of humanization is a reduction in the immunogenicity of axenogenic antibody, such as a murine antibody, for introduction into ahuman, while maintaining the full antigen binding affinity andspecificity of the antibody.

Humanized antibodies may be produced using several technologies such asresurfacing and CDR grafting. As used herein, the resurfacing technologyuses a combination of molecular modeling, statistical analysis andmutagenesis to alter the non-CDR surfaces of antibody variable regionsto resemble the surfaces of known antibodies of the target host.

Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641 (Pedersen et al.), whichis hereby incorporated in its entirety by reference. Briefly, in apreferred method, (1) position alignments of a pool of antibody heavyand light chain variable regions is generated to give a set of heavy andlight chain variable region framework surface exposed positions whereinthe alignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a rodent antibody (orfragment thereof); (3) a set of heavy and light chain variable regionframework surface exposed amino acid residues that is most closelyidentical to the set of rodent surface exposed amino acid residues isidentified; (4) the set of heavy and light chain variable regionframework surface exposed amino acid residues defined in step (2) issubstituted with the set of heavy and light chain variable regionframework surface exposed amino acid residues identified in step (3),except for those amino acid residues that are within 5 Å of any atom ofany residue of the complementarity-determining regions of the rodentantibody; and (5) the humanized rodent antibody having bindingspecificity is produced.

Antibodies can be humanized using a variety of other techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., 1991, Molecular Immunology 28(4/5):489-498;Studnicka G. M. et al., 1994, Protein Engineering 7(6):805-814; RoguskaM. A. et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Human antibodies can be made by a variety of methods knownin the art including phage display methods. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patentapplication publication numbers WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said referencesincorporated by reference in their entireties).

As further described herein, the CDRs of My9-6 were identified bymodeling and their molecular structures were predicted. Humanized My9-6antibodies were then prepared and have been fully characterized. Theamino acid sequences of the light and heavy chains of a number ofhuMy9-6 antibodies are shown in FIGS. 16A and 16B. Comparative bindingvalues for murine and humanized My9-6 antibodies are provided in FIG.17. Binding curves for the antibodies are shown in FIG. 18.

Epitope-Binding Fragments of the My9-6 Antibodies

Although epitope-binding fragments of the murine My9-6 antibody and thehumanized My9-6 antibodies are discussed herein separately from themurine My9-6 antibody and the humanized versions thereof, it isunderstood that the term “antibody” or “antibodies” of the presentinvention may include both the full length muMy9-6 and huMy9-6antibodies as well as epitope-binding fragments of these antibodies.

As used herein, “antibody fragments” include any portion of an antibodythat retains the ability to bind to CD33, generally termed“epitope-binding fragments.” Examples of antibody fragments preferablyinclude, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd, single-chainFvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) andfragments comprising either a V_(L) or V_(H) domain. Epitope-bindingfragments, including single-chain antibodies, may comprise the variableregion(s) alone or in combination with the entirety or a portion of thefollowing: hinge region, CH₁, CH₂, and CH₃ domains.

Such fragments may contain one or both Fab fragments or the F(ab′)₂fragment. Preferably, the antibody fragments contain all six CDRs of thewhole antibody, although fragments containing fewer than all of suchregions, such as three, four or five CDRs, are also functional. Further,the functional equivalents may be or may combine members of any one ofthe following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, andthe subclasses thereof.

Fab and F(ab′)₂ fragments may be produced by proteolytic cleavage, usingenzymes such as papain (Fab fragments) or pepsin (F(ab′)₂ fragments).

The single-chain FVs (scFvs) fragments are epitope-binding fragmentsthat contain at least one fragment of an antibody heavy chain variableregion (V_(H)) linked to at least one fragment of an antibody lightchain variable region (V_(L)). The linker may be a short, flexiblepeptide selected to assure that the proper three-dimensional folding ofthe (V_(L)) and (V_(H)) regions occurs once they are linked so as tomaintain the target molecule binding-specificity of the whole antibodyfrom which the single-chain antibody fragment is derived. The carboxylterminus of the (V_(L)) or (V_(H)) sequence may be covalently linked bya linker to the amino acid terminus of a complementary (V_(L)) and(V_(H)) sequence. Single-chain antibody fragments may be generated bymolecular cloning, antibody phage display library or similar techniqueswell known to the skilled artisan. These proteins may be produced, forexample, in eukaryotic cells or prokaryotic cells, including bacteria.

The epitope-binding fragments of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In particular, such phage can be utilized to displayepitope-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing anepitope-binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled CD33 or CD33bound or captured to a solid surface or bead. Phage used in thesemethods are typically filamentous phage including fd and M13 bindingdomains expressed from phage with Fab, Fv or disulfide-stabilized Fvantibody domains recombinantly fused to either the phage gene III orgene VIII protein.

Examples of phage display methods that can be used to make theepitope-binding fragments of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ameset al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al.,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;Burton et al., 1994, Advances in Immunology 57:191-280; PCT applicationNo. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108; each of which is incorporated hereinby reference in its entirety.

After phage selection, the regions of the phage encoding the fragmentscan be isolated and used to generate the epitope-binding fragmentsthrough expression in a chosen host, including mammalian cells, insectcells, plant cells, yeast, and bacteria, using recombinant DNAtechnology, e.g., as described in detail below. For example, techniquesto recombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., 1992, BioTechniques12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043; said references incorporated by referencein their entireties. Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology203:46-88; Shu et al., 1993, PNAS 90:7995-7999; Skerra et al., 1988,Science 240:1038-1040.

Functional Equivalents

Also included within the scope of the invention are functionalequivalents of the My9-6 antibody and the humanized My9-6 antibodies.The term “functional equivalents” includes antibodies with homologoussequences, chimeric antibodies, modified antibody and artificialantibodies, for example, wherein each functional equivalent is definedby its ability to bind to CD33. The skilled artisan will understand thatthere is an overlap in the group of molecules termed “antibodyfragments” and the group termed “functional equivalents.”

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence identity or homology with amino acidsequence of the murine My9-6 and humanized My9-6 antibodies of thepresent invention. Preferably identity is with the amino acid sequenceof the variable regions of the murine My9-6 and humanized My9-6antibodies of the present invention. “Sequence identity” and “sequencehomology” as applied to an amino acid sequence herein is defined as asequence with at least about 90%, 91%, 92%, 93%, or 94% sequenceidentity, and more preferably at least about 95%, 96%, 97%, 98%, or 99%sequence identity to another amino acid sequence, as determined, forexample, by the FASTA search method in accordance with Pearson andLipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).

As used herein, a chimeric antibody is one in which different portionsof an antibody are derived from different animal species. For example,an antibody having a variable region derived from a murine monoclonalantibody paired with a human immunoglobulin constant region. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies etal., 1989, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties.

Artificial antibodies include scFv fragments, diabodies, triabodies,tetrabodies and mru (see reviews by Winter, G. and Milstein, C., 1991,Nature 349: 293-299; Hudson, P. J., 1999, Current Opinion in Immunology11: 548-557), each of which has antigen-binding ability. In the singlechain Fv fragment (scFv), the V_(H) and V_(L) domains of an antibody arelinked by a flexible peptide. Typically, this linker peptide is about 15amino acid residues long. If the linker is much smaller, for example 5amino acids, diabodies are formed, which are bivalent scFv dimmers. Ifthe linker is reduced to less than three amino acid residues, trimericand tetrameric structures are formed that are called triabodies andtetrabodies. The smallest binding unit of an antibody is a CDR,typically the CDR2 of the heavy chain which has sufficient specificrecognition and binding that it can be used separately. Such a fragmentis called a molecular recognition unit or mru. Several such mrus can belinked together with short linker peptides, therefore forming anartificial binding protein with higher avidity than a single mru.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Thecovalent attachment does not prevent the antibody from generating ananti-idiotypic response. These modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the modified antibodies may contain one or morenon-classical amino acids.

Functional equivalents may be produced by interchanging different CDRson different chains within different frameworks. Thus, for example,different classes of antibody are possible for a given set of CDRs bysubstitution of different heavy chains, whereby, for example, IgG₁₋₄,IgM, IgA₁₋₂, IgD, IgE antibody types and isotypes may be produced.Similarly, artificial antibodies within the scope of the invention maybe produced by embedding a given set of CDRs within an entirelysynthetic framework.

Functional equivalents may be readily produced by mutation, deletionand/or insertion within the variable and/or constant region sequencesthat flank a particular set of CDRs, using a wide variety of methodsknown in the art.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of bindingto CD33, when compared to the murine My9-6 antibody. A detectable degreeof binding includes all values in the range of at least 10-100%,preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%,85%, 90%, 95% or 99% the binding ability of the murine My9-6 antibody toCD33.

Improved Antibodies

The CDRs are of primary importance for epitope recognition and antibodybinding. However, changes may be made to the residues that comprise theCDRs without interfering with the ability of the antibody to recognizeand bind its cognate epitope. For example, changes that do not affectepitope recognition, yet increase the binding affinity of the antibodyfor the epitope may be made.

Thus, also included in the scope of the present invention are improvedversions of both the murine and humanized antibodies, which alsospecifically recognize and bind CD33, preferably with increasedaffinity.

Several studies have surveyed the effects of introducing one or moreamino acid changes at various positions in the sequence of an antibody,based on the knowledge of the primary antibody sequence and on itsproperties such as binding and level of expression (Yang, W. P. et al.,1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Natl.Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et al., 1998, NatureBiotechnology, 16, 535-539).

In these studies, equivalents of the primary antibody have beengenerated by changing the sequences of the heavy and light chain genesin the CDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resultedin improved affinities of the secondary antibodies (Gram, H. et al.,1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder, E. T. et al.,2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. andRiechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et al.,1996, J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277, 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276,27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described herein can beused to develop anti-CD33 antibodies with improved functions, includingimproved affinity for CD33.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics

Immunoconjugates

The present invention is also directed to immunoconjugates, comprisingthe antibodies, antibody fragments, functional equivalents, improvedantibodies and their analogs as disclosed herein, linked to a drug orprodrug. Preferred drugs or prodrugs are cytotoxic agents and include,for example, maytansinoids and maytansinoid analogs, taxoids, CC-1065and CC-1065 analogs, dolastatin and dolastatin analogs.

The immunoconjugates may be prepared by in vitro methods. In order tolink a drug or prodrug to the antibody, a linking group is used.Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred linkinggroups are disulfide groups and thioether groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between the antibody and the drug or prodrug.

Maytansinoids and maytansinoid analogs are among the preferred cytotoxicagents. Examples of suitable maytansinoids include maytansinol andmaytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat.Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

With respect to maytansinoids, the linking group may comprise a reactivechemical group. In a preferred embodiment, the reactive chemical groupcan be covalently bound to the maytansinoid via a disulfide bond linkingmoiety.

Particularly preferred reactive chemical groups are N-succinimidylesters and N-sulfosuccinimidyl esters.

Particularly preferred maytansinoids comprising a linking group thatcontains a reactive chemical group are C-3 esters of maytansinol and itsanalogs where the linking moiety contains a disulfide bond and thechemical reactive group comprises a N-succinimidyl orN-sulfosuccinimidyl ester.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. However the C-3 position is preferred and theC-3 position of maytansinol is especially preferred.

Other chemical bonds include acid labile bonds, photolabile bonds,peptidase labile bonds and esterase labile bonds. The disclosure of U.S.Pat. No. 5,208,020, incorporated herein, teaches the production ofmaytansinoids bearing such bonds.

As described in detail below, the immunocojugate My9-6-DM1 utilizesthiol-containing maytansinoid (DM1). DM1 is represented by the followingstructural formula (I):

Taxanes are also preferred cytotoxic agents. Taxanes suitable for use inthe present invention are disclosed in U.S. Pat. Nos. 6,372,738 and6,340,701. Conjugates of the taxanes of the invention and a cell bindingagent can be formed using any techniques presently known or laterdeveloped. Numerous methods of conjugation are taught in U.S. Pat. No.5,416,064 and U.S. Pat. No. 5,475,092.

CC-1065 and its analogs are also preferred cytotoxic drugs for use inthe present invention. CC-1065 and its analogs are disclosed in U.S.Pat. Nos. 6,372,738; 6,340,701; 5,846,545; and 5,585,499. CC-1065 is apotent anti-tumor antibiotic isolated from the culture broth ofStreptomyces zelensis. CC-1065 is about 1000-fold more potent in vitrothan commonly used anti-cancer drugs, such as doxorubicin, methotrexateand vincristine (B. K. Bhuyan et al., Cancer Res., 42, 3532-3537(1982)).

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin,dolastatin and dolastatin analogs are also suitable for the preparationof conjugates of the present invention. The drug molecules can also belinked to the antibody molecules through an intermediary carriermolecule such as serum albumin.

Inhibiting the Growth of CD33-Expressing Cells

Also included in the present invention are methods for inhibiting thegrowth of cells expressing CD33. These methods make use of theantibodies or immunoconjugates of the present invention, as well as theantibodies or immunoconjugates of the present invention in conjunctionwith one or more additional therapeutic agents. Suitable therapeuticagents include those that inhibit the growth of a cell expressing CD33directly or indirectly.

As used herein the terms “inhibit” and “inhibiting” should be understoodto include any inhibitory effect on cell growth, including cell death.The inhibitory effects include temporary effects, sustained effects andpermanent effects.

Therapeutic Applications

The present invention also includes therapeutic applications of theantibodies or immunoconjugates of the present invention wherein theantibodies or immunoconjugates may be administered to a subject, in apharmaceutically acceptable dosage form. They can be administeredintravenously as a bolus or by continuous infusion over a period oftime, by intramuscular, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. They may also beadministered by intratumoral, peritumoral, intralesional, orperilesional routes, to exert local as well as systemic therapeuticeffects.

A pharmaceutically acceptable dosage form will generally include apharmaceutically acceptable agent such as a carrier, diluent, andexcipient. These agents are well known and the most appropriate agentcan be determined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH ˜7.4, containingabout 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/vNaCl), and (3) 5% (w/v) dextrose.

In other therapeutic applications, the antibodies or immunoconjugates ofthe invention are co-administered with one or more additionaltherapeutic agents. Therapeutic agents are those agents that seek tokill or limit the growth of cancer cells while doing minimal damage tothe host. Thus, such agents may exploit any difference in cancer cellproperties (e.g. metabolism, vascularization or cell-surface antigenpresentation) from healthy host cells. Differences in tumor morphologyare potential sites for intervention. For example, the therapeutic agentcan be an antibody such as an anti-VEGF antibody that is useful inretarding the vascularization of the interior of a solid tumor, therebyslowing its growth rate.

Suitable therapeutic agents include, but are not limited to, cytotoxicor cytostatic agents. Taxol is a preferred therapeutic agent that isalso a cytotoxic agent.

Other therapeutic agents include, but are not limited to, adjuncts suchas granisetron HCL, androgen inhibitors such as leuprolide acetate,antibiotics such as doxorubicin, antiestrogens such as tamoxifen,antimetabolites such as interferon alpha-2a, enzyme inhibitors such asras farnesyl-transferase inhibitor, immunomodulators such asaldesleukin, and nitrogen mustard derivatives such as melphalan HCl, andthe like.

When present in an aqueous dosage form, rather than being lyophilized,the antibody typically will be formulated at a concentration of about0.1 mg/ml to 100 mg/ml, although wide variation outside of these rangesis permitted. For the treatment of disease, the appropriate dosage ofantibody or conjugate will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether theantibodies are administered for preventive or therapeutic purposes, thecourse of previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 0.015 to 15 mgof antibody/kg of patient weight is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and arenot excluded.

The therapeutic applications of the present invention include methods oftreating a subject having a disease. The diseases treated with themethods of the present invention are those characterized by theexpression of CD33. Such diseases include myelodysplastic syndromes(MDS) and cancers such as acute myeloid leukemia (AML), chronic myeloidleukemia (CML) and pro-myelocytic leukemia (PML). The skilled artisanwill understand that the methods of the present invention may also beused to treat other diseases yet to be described but characterized bythe expression of CD33.

The therapeutic applications of the present invention can be alsopracticed in vitro and ex vivo.

Examples of in vitro uses include the purification of cell populationscontaminated with CD33-positive cells such as cells of myeloid lineage.The method comprises culturing the cell populations in the presence of acytotoxic My9-6 immunoconjugate and then removal of dead, CD33-positivecells. The conditions for non-clinical in vitro use are well known (see,e.g., Uckun et al., 1986, J Exp. Med. 163, 347-368; Uckun et al., 1985,J. Immunol. 134, 3504-3515; Ramakrishnan et al., 1985, J. Immunol.3616-3622).

Examples of clinical ex vivo use include treatment of autologous bonemarrow prior to their infusion into the same patient in order to killdiseased or malignant myeloid lineage cells (Roy D. C. et al., 1995, J.Clin. Immunol. 15, 51-57).

Diagnostic and Research Applications

In addition to the therapeutic uses of the antibodies discussed herein,the antibodies of the present invention can be employed in many knowndiagnostic and research applications. Antibodies of the presentinvention may be used, for example, in the purification, detection, andtargeting of CD33, included in both in vitro and in vivo diagnosticmethods. For example, the antibodies may be used in immunoassays forqualitatively and quantitatively measuring levels of CD33 expressed bycells in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988),incorporated by reference herein in its entirety.

The antibodies of the present invention may be used in, for example,competitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent or radioisotope is administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled antibody in the host is assayed. This imaging technique isuseful in the staging and treatment of malignancies. The antibody may belabeled with any moiety that is detectable in a host, whether by nuclearmagnetic resonance, radiology, or other detection means known in theart.

The label can be any detectable moiety that is capable of producing,either directly or indirectly, a detectable signal. For example, thelabel may be a biotin label, an enzyme label (e.g., luciferase, alkalinephosphatase, beta-galactosidase and horseradish peroxidase), aradio-label (e.g., ³H, ¹⁴C, ³²P, ³⁵S, and ¹²⁵I), a fluorophore such asfluorescent or chemiluminescent compound (e.g., fluoresceinisothiocyanate, rhodamine), an imaging agent (e.g., Tc-m99 and indium(¹¹¹In)) and a metal ion (e.g., gallium and europium).

Any method known in the art for conjugating the antibody to the labelmay be employed, including those methods described by Hunter, et al.,1962, Nature 144:945; David et al., 1974, Biochemistry 13:1014; Pain etal., 1981, J. Immunol. Meth. 40:219; Nygren, J., 1982, Histochem. andCytochem. 30:407.

The antibodies of the invention also are useful as affinity purificationagents. In this process, the antibodies are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. Thus, CD33 may be isolated and purified from a biologicalsample.

Polynucleotides, Vectors, Host Cells and Methods for Making Antibody

The present invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention orepitope-binding fragments thereof.

The present invention also encompasses polynucleotides encoding apolypeptide that can bind CD33 and that hybridize under stringenthybridization conditions to polynucleotides that encode an antibody ofthe present invention, wherein said stringent hybridization conditionsinclude: pre-hybridization for 2 hours at 60° C. in 6×SSC, 0.5% SDS,5×Denhardt's solution, and 100 μg/ml heat denatured salmon sperm DNA;hybridization for 18 hours at 60° C.; washing twice in 4×SSC, 0.5% SDS,0.1% sodium pyrophosphate, for 30 min at 60° C. and twice in 2×SSC, 0.1%SDS for 30 min at 60° C.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242) which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligation of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Methods for the construction of recombinant vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals are well known in the art. These methods include, forexample, in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. The invention, thus, provides replicablevectors comprising a nucleotide sequence encoding an antibody moleculeof the present invention, or a heavy or light chain thereof, or a heavyor light chain variable domain, or an epitope-binding fragment of any ofthese, operably linked to a promoter.

The recombinant vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or an epitope-binding fragment thereof, operably linkedto a heterologous promoter. In preferred embodiments, vectors encodingboth the heavy and light chains may be co-expressed in the host cell forexpression of an entire immunoglobulin molecule.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

Preferably, bacterial cells such as Escherichia coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.) and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention.

Example 1 Murine My9-6 Antibody

In this first example, the complete primary amino acid structure andcDNA sequence of the murine My9-6 antibody of the present invention isdisclosed, together with its binding properties and means for itsexpression in recombinant form. Accordingly, there is provided a fulland complete disclosure of an antibody of the invention and itspreparation, such that one of ordinary skill in the immunological artswould be able to prepare said antibody without undue experimentation.

1.1. Generation, Production and Characterization of My9-6 Antibody

A 3T3 murine fibroblast cell line transfected with the CD33 antigen wasused for immunization.

BALB/c female mice, 5 months old, were immunized intraperitoneally onday 0 with the transfected 3T3 murine fibroblast cell line (2.5×10⁶cells, suspended in 0.2 mL PBS). The animals were boosted with 0.2 mLcell suspension as follows: day 13, 5×10⁶ cells; day 21, 5×10⁶ cells. Onday 24, a mouse was sacrificed and its spleen removed.

The spleen was ground between two frosted glass slides to obtain asingle cell suspension, which was washed with serum-free RPMI mediumcontaining penicillin and streptomycin (SFM). The spleen cell pellet wasresuspended in 10 mL of 0.83% (w/v) ammonium chloride solution in waterfor 10 min on ice to lyse the red blood cells, and was then washed withserum-free medium (SFM). Spleen cells (1.6×10⁸) were pooled with myelomacells (5.4×10⁷) from the non-secreting mouse myeloma cell lineP3X63Ag8.653 (ATCC, Rockville, Md.; cat. no. CRL1580) in a tube, andwashed with the serum-free RPMI-1640 medium (SFM). The supernatant wasremoved and the cell pellet diluted to 2×10⁷ cells/mL. The cells wereplated at 2×10⁷ cells/plate onto tissue culture plates that were coatedwith 15 mg/mL concanavalin A. Plates were incubated for one hour at 37°C. The supernatant was gently removed from the plates. One mL ofpolyethylene glycol solution (40% PEG w/v) was slowly added dropwise.The plates were swirled and incubated for 30 seconds. The PEG wasremoved and discarded. The plates were washed twice by slowly adding 5mL of SFM and then discarding. After the final wash, 5 mL of SFMsupplemented with 5% Fetal Bovine Serum (FBS) was added. Plates wereincubated overnight at 37° C. Following incubation, cells were scrapedoff the plates with a cell scraper and pooled. The plates were rinsedand pooled with the cells. Cells were pelleted by centrifugation andresuspended in RPMI-1640 growth media supplemented with 5% FBS,penicillin-streptomycin and hypoxanthine/aminopterin/thymidine (HAT).Cells were plated at 2×10⁵ cells/well on 96-well flat bottom tissueculture plates containing a macrophage feeder layer. The generalconditions used for immunization and hybridoma production were asdescribed by J. Langone and H. Vunakis (Eds., Methods in Enzymology,Vol. 121, “Immunochemical Techniques, Part I”; 1986; Academic Press,Florida) and E. Harlow and D. Lane (“Antibodies: A Laboratory Manual”;1988; Cold Spring Harbor Laboratory Press, New York). Other techniquesof immunization and hybridoma production can also be used, as are wellknown to those of skill in the art.

Culture supernatants from hybridoma clones were screened for binding tocells transfected with the CD33 antigen and the human histiocyticlymphoma cell line, U-937 (ATCC CRL-1953.2) and for a lack of binding toa mouse fibroblast cell line. These clones were expanded and subcloned.The culture supernatants of the subclones were further screened by theabove binding assays. By this procedure, subclone 3E7-H2-3D8 (My9-6) wasselected, and the heavy and light chain genes were cloned and sequencedas described below.

Screening of hybridoma supernatants for specific binding to the CD33antigen was performed using ELISA on cell lines that expressed thisantigen and on a cell line negative for this antigen. Cells wereseparately harvested from tissue culture flasks, suspended in growthmedium containing 10% FBS, pelleted by centrifugation, and washed withPBS. The washed cells (100 μL of about 1-3×10⁶ cells/mL) were added towells of an Immulon-2HB plate coated with phytohemagglutinin (100 μL of20 μg/mL PHA), centrifuged and allowed to adhere to PHA-coated wells for10 min. The plate with cells was flicked to remove PBS and was thendried overnight at 37° C. The wells were blocked with 5 mg/mL BSAsolution in PBS for 1 h at 37° C. and were then washed gently with PBS.Aliquots of the supernatants from hybridoma clones (100 μL; diluted inblocking buffer) were then added to wells containing CD33antigen-expressing cells and to cells not expressing CD33, and wereincubated at ambient temperature for 1 h. The wells were washed withPBS, incubated with goat-anti-mouse-IgG-antibody-horseradish peroxidaseconjugate (100 μL; in blocking buffer) for 1 h, followed by washes andthen binding was detected using an ABTS/H₂O₂ substrate. A typicalsupernatant from a 3E7 hybridoma subclone upon incubation with cellsoverexpressing CD33 antigen yielded a signal of 0.50 absorbance units,in contrast to a value of 0.10 absorbance units obtained upon incubationwith cells negative for the CD33 antigen.

The hybridoma was grown in ascites in BALB/c mice. A vial of frozenhybridoma cells was thawed and the cells were expanded in tissue cultureflasks to obtain the necessary number of cells for ascites production.Primed BALB/c mice (mice had been injected i.p. with 0.5 mL of pristane10-14 days in advance) were injected i.p. with 1×10⁶ cells in 0.5 mL ofphosphate buffered saline (PBS). Twelve to 18 days after injection ofthe cells, ascites fluid was withdrawn from the peritoneal cavity of themice with a syringe. The pooled ascites fluid was centrifuged at 1000rpm for 5 min, the supernatant was then subjected to centrifugation at12,000 rpm for 30 minutes. The antibody was purified from the clearsupernatant as follows: Step 1: ammonium sulfate precipitation. Thesupernatant on ice was diluted with two volumes of cold PBS, stirred andthen treated slowly with one volume of cold, saturated (100%) ammoniumsulfate solution. The solution was left standing on ice for about 5hours, when the precipitate was collected by centrifugation. The pelletwas dissolved in a small amount of PBS and the resulting solution wasdialyzed into the buffer for the affinity purification step with proteinA. Step 2: Affinity purification on Sepharose-Protein A. The isotype ofmurine My9-6 is IgG1 with a kappa light chain. Therefore the affinitycolumn was equilibrated in 0.1 M tris.HCl buffer containing 3 M NaCl, pH8.0. The antibody solution in the same buffer was passed through thecolumn, the column was washed well with the equilibrating buffer andthen eluted with 0.1 M acetic acid containing 0.15 M NaCl. Fractionswere assayed by measuring the UV absorption at 280 nm. Fractionscontaining the antibody were combined, neutralized with 1 M tris andthen dialyzed into PBS for use and storage.

Purified antibody was initially characterized for binding toCD33-expressing cells, such as the transfected murine 3T3 fibroblastcell line and the human U-937 cell line, and the absence of binding toantigen-negative cell lines as described above for the screening ofhybridoma supernatants.

To further ascertain its specificity for binding to the CD33 antigen, acompetition binding experiment was performed between labeled muMy9-6 andthe commercially available anti-CD33 antibody My9. The bindingexperiment was performed using the method described by Goldmacher et al.(1989, J. Cell. Physiol. 141, 222-234). Murine My9-6 antibody wasradiolabeled with ¹²⁵I using the Iodo-gen technique (Fraker, P. J. andSpeck, J. C., 1978, Biochem. Biophys. Res. Commun. 80, 849-857). Aconstant amount (3×10⁻⁹ M) of ¹²⁵I-labeled muMy9-6 was mixed withincreasing concentrations of non-radioactive antibody, either unlabeledmuMy9-6 or unlabelled My9, and the mixtures were incubated with cells(1×10⁶ cells per sample) at 4° C. for 30 min. These incubations weredone in 60 μl of a buffer consisting of Minimum Essential MediumModified for Suspension Cultures (SMEM) supplemented with 2.5% humanserum. The cells were then separated from the media containing non-boundmaterial by centrifugation through a mixture of silicone oil (Aldrich)and paraffin oil (Baker) with a density of 1.009 g/ml. The cell pelletwas then removed by cutting the centrifuge tubes at the oil interfaceand was used to measure the cell-associated radioactivity on agamma-counter (model RIAgamma 1274 from LKB). The cell-associatedradioactivity measured was blotted against the concentrations of theunlabelled, competing antibodies. The results are shown in FIG. 1: bothantibodies, My9 and My9-6, give very similar competition binding curves.This demonstrates that My9 and My9-6 bind to the same antigen andfurthermore that both antibodies have very similar binding avidities.Since My9 is a published and accepted anti-CD33 standard it wasconcluded that My9-6 is an anti-CD33 antibody. My9 antibody iscommercially available from BioGenex (San Ramon, Calif. 94583; cat. no.AM267-5M).

1.2. Cloning of Heavy and Light Chain Genes of My9-6 Antibody

Total RNA was purified from a confluent T175 flask of My9-6 hybridomacells as described in Molecular Protocols (NB1455-p173). RNA integritywas checked on a 1% MOPS gel and concentrations were determined by UVspectrophotometry. RT reactions were done with 4-5 μg total RNA usingthe Gibco Superscript II kit and either oligo dT or random hexamerprimers.

PCR reactions were done both by using a RACE method described in Co etal. (1992, J. Immunol. 148(4):1149-54) and by using degenerate primersdescribed in Wang et al. (2000, J. Immunol. Methods. 233:167-177). TheRACE PCR required an intermediate step to add a poly dG tail on the 3′ends of the first strand cDNAs. RT reactions were purified with Qianeasy(Qiagen) columns and eluted in 50 μl 1×NEB buffer 4. A dG tailingreaction was done on the eluate with 0.25 mM CoCl₂, 1 mM dGTP, and 5units terminal transferase (NEB), in 1×NEB buffer 4. The mixture wasincubated at 37° C. for 30 minutes and then ⅕ of the reaction mixture(10 μl) was added directly to a PCR reaction mixture to serve as thetemplate DNA.

The RACE and degenerate PCR reactions were identical except fordifferences in primers and template. As mentioned above, the terminaltransferase reaction mixture was used directly for the RACE PCRtemplate, while the RT reaction mix was used directly for degenerate PCRreactions. In both reaction sets the same 3′ light chain primer, HindKL:

(SEQ ID NO: 11) TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGCand 3′ heavy chain primer, Bg12IgG1:

(SEQ ID NO: 12) GGAAGATCTATAGACAGATGGGGGTGTCGTTTTGGCwere used.

In the RACE PCR, one poly dC 5′ primer was used for both the heavy andlight chain, EcoPolydC: TATATCTAGAATTCCCCCCCCCCCCCCCCC (SEQ ID NO:13),while the degenerate 5′ end PCR primers were Sac1MK:GGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO:14) for the light chain andan equal mix of EcoR1MH1: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ IDNO:15) and EcoR1MH2: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO:16)for the heavy chain (mixed bases: H=A+T+C, S=G+C, Y=C+T, K=G+T, M=A+C,R=A+G, W=A+T, V=A+C+G, N=A+T+G+C).

The PCR reactions were performed on an MJ research thermocycler using aprogram adapted from Wang et al. (2000, J. Immunol. Methods.233:167-177): 1) 94° C., 3 min; 2) 94° C., 15 sec; 3) 45° C., 1 min; 4)72° C., 2 min; 5) cycle back to step #2 29 times; 6) finish with a finalextension step at 72° C. for 10 min. The PCR products were cloned intopBluescript II SK+ (Stratagene) using restriction enzymes created by thePCR primers. Heavy and light chain clones were sequenced by LarkTechnologies or Seqwright sequencing services.

The RACE PCR was never successful for the My9-6 light chain. Thus, inorder to confirm the 5′ end cDNA sequences, additional degenerate PCRand cloning was done. The My9-6 light chain cDNA sequence, determinedfrom the degenerate PCR clones, was entered into the NCBI's Blast searchwebsite (ncbi.nlm.nih.gov/BLAST/) and the top five murine antibodysequences with signal sequence submitted were saved. Degenerate PCRprimers were designed from these signal peptides using the Codehop websoftware (blocks.fhcrc.org/codehop.html). EcoRI restriction sites wereadded to three of the Codehop degenerate primers (FIG. 2) and these wereused in RT-PCR reactions as described above.

1.3. Preparation and Sequencing of Heavy and Light Chain Samples

The heavy and light chains of muMy9-6 antibody were separated bySDS-PAGE under reducing conditions. The reduced and denatured antibodywas electrophoresed on a 12% Tris-glycine gel (Novex, San Diego,Calif.). After electrophoresis, the gels were blotted onto anImmobilon^(psq) membrane using CAPS/MeOH buffer. After transfer, themembranes were stained with Ponseau S. The bands corresponding to thelight and heavy chains were excised for protein sequencing.

The light chain of the antibody was sequenced directly from the membraneby automated Edman degradation chemistry on an ABI 494 Procisesequencer.

The N-terminus of the heavy chain was blocked, thus the protein wasdigested, in situ, with trypsin according to Gharahdaghi, et al. (1996).The digest mixture was then analyzed by MALDI-TOF mass spectrometry on aKratos Kompact SEQ instrument. Selected peptides were subjected to MS/MSto determine their sequences.

Example 2 Humanization of Antibody Variable Region by Resurfacing

Resurfacing of the My9-6 antibody to provide humanized versions suitableas therapeutic or diagnostic agents generally proceeds according to theprinciples and methods disclosed in U.S. Pat. No. 5,639,641, and asfollows.

2.1. Surface Prediction The solvent accessibility of the variable regionresidues for a set of antibodies with solved structures was used topredict the surface residues for the murine My9-6 antibody variableregion. The amino acid solvent accessibility for a set of 127 uniqueantibody structure files (FIG. 3) were calculated with the MC softwarepackage (Pedersen et al., 1994, J. Mol. Biol. 235(3):959-973). The tenmost similar light chain and heavy chain amino acid sequences from thisset of 127 structures was determined using sequence alignment softwareon the NCBI website ncbi.nlm.nih.gov/BLAST/. The average solventaccessibility for each variable region residue of these ten antibodyvariable regions was calculated with an Excel spreadsheet and positionswith greater than a 30% average accessibility were considered surfaceresidues. Positions with average accessibilities of between 25% and 35%were further considered by calculating the individual residueaccessibility for only those structures with two identical residuesflanking on either side.

2.2. Molecular Modeling

A molecular model of the variable region of murine My9-6 was generatedusing the Oxford Molecular software package AbM. The antibody frameworkwas built from structure files for the antibodies with the most similaramino acid sequences (1sbs for the light chain and 1bbj for the heavychain) and the non-canonical CDRs were built by searching a C-αstructure database containing non-redundant solved structures. Modelswere viewed with the GlaxoSmithKline Swiss-Pdb Viewer and residues thatlie within 5 Å of a CDR were determined.

2.3. Human Antibody Selection

The surface positions of the murine My9-6 variable region were comparedto the corresponding positions in human antibody sequences in the Kabatdatabase (Johnson and Wu, 2001, Nucleic Acids Res. 29(1):205-6). Theantibody database management software SR (Searle, 1998) was used toextract and align the surface residues from natural heavy and lightchain human antibody pairs. The human antibody variable region surfacewith the most identical surface residues, with special considerationgiven to positions that come within 5 Å of a CDR, was chosen to replacethe murine My9-6 antibody variable region surface residues.

2.4. Construction of Humanized Antibody Gene by PCR Mutagenesis

PCR mutagenesis was performed on the murine My9-6 variable region cDNAclones to first change the 5′ end sequences to match the peptidesequence, and then build the resurfaced, human My9-6 gene. Primer setswere designed to create the murine residues not originally sequenced inthe initial degenerate PCR clones (light chain positions N1, M3, L4, andS7, and heavy chain positions Q3 and P7).

Humanization primer sets were designed to make the 6 amino acid changesrequired for all versions of huMy9-6 and additional primers weredesigned to alternatively change the four 5 Å residues (FIG. 4). PCRreactions were performed on an MJ Research thermocycler with thefollowing program: 1) 94° C., 1 min; 2) 94° C., 15 sec; 3) 55° C., 1min; 4) 72° C., 1 min; 5) cycle back to step #2 29 times; 6) finish witha final extension step at 72° C. for 4 min. The PCR products weredigested with their corresponding restriction enzymes and cloned intothe pBluescript cloning vectors. Clones were sequenced to confirm theamino acid changes.

2.5. Expression Vector for Humanized Antibodies

The light and heavy chain paired sequences were cloned into a singlemammalian expression vector. The PCR primers for the human variablesequences created restriction sites that allowed the human signalsequence to be added in the pBluescriptII cloning vector. The variablesequences could then be cloned into the mammalian expression plasmidwith EcoRI and BsiWI or HindIII and ApaI for the light chain or heavychain respectively (FIG. 5). The light chain variable sequences werecloned in-frame onto the human IgKappa constant region and the heavychain variable sequences were cloned into the human IgGamma1 constantregion sequence. In the final expression plasmids, human CMV promotersdrive the expression of both the light and heavy chain cDNA sequences.

2.6. Transient Expression of Humanized Antibodies

293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM,BioWhittaker, 12-614F), 10% heat-inactivated fetal bovine serum(Hyclone, SH30071.03), 4 mM L-glutamine (BioWhittaker, 17-604E), and 1%penicillin/streptomycin mixture (BioWhittaker, 17-603E) under 6% CO₂ ina 37° C. incubator. The cells were split three times per week at a 1:10dilution maintaining a sub-confluent population. 24 hours prior totransfection, cells were trypsinized (BioWhittaker, 17-161E); live cellswere counted by the trypan blue exclusion method and plated on 10 cm,tissue culture treated plates (Corning, 430167) at a density of 2×10⁶cells per plate. Immediately prior to transfection, cells were washedgently with phosphate buffered saline (PBS, diluted from BioWhittaker10×PBS, 17-517Q) and cells were overlayed with 7 mL of Hybridoma SFM(InvitroGen, 12045-076) including 1% Ultra Low IgG Serum (Gibco BRL,16250-078).

The transient transfection methods were adapted from the standard QiagenPolyfect protocol. For one 10 cm plate to be transfected, 8 μg of CsClgrade DNA was combined with 300 μL Hybridoma SFM and 80 μL Polyfecttransfection reagent (Qiagen, 301107). The transfection mix was vortexedfor about 5 seconds on low speed and incubated for 5 minutes at ambienttemperature. After incubation, 1 mL of Hybridoma SFM, 1% Ultra Low IgGSerum was added to the transfection mix and combined by pipetting up anddown about five times. Transfection mix was then added to the 7 mLHybridoma SFM covering the cells and plates were swirled gently toinsure even distribution. The transfection reactions were incubatedover-night, generally 16 hours, in a tissue culture incubator. Thetransfection media was then carefully removed from the cells andreplaced with 20 mL of Hybridoma SFM, 1% Ultra Low IgG. The transfectedcells were then returned to the incubator for 72 hours, after which thesupernatants were harvested and antibody production quantitated byELISA. Harvested supernatant was stored at 4° C. until purification.

2.7. Purification of Humanized Antibodies

Supernatant was prepared for Protein A affinity chromatography by theaddition of 1/10 volume of 1 M Tris/HCl buffer, pH 8.0. The pH-adjustedsupernatant was filtered through a 0.22 μm filter membrane and loadedonto a Protein A Sepharose column (HiTrap Protein A HP, 1 mL, AmershamBiosciences) equilibrated with binding buffer (PBS, pH 7.3). AQ-Sepharose precolumn (10 mL) was connected upstream of the Protein Acolumn during sample loading to reduce contamination from cellularmaterial such as DNA. Following sample loading, the pre-column wasremoved and the Protein A column orientation was reversed for wash andelution. The column was washed with binding buffer until a stablebaseline was obtained with no absorbance at 280 nm. Antibody was elutedwith 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, using aflow rate of 0.5 mL/min. Fractions of approximately 0.25 mL werecollected and neutralized by the addition of 1/10 volume of 1M Tris/HCl,pH 8.0. The peak fraction(s) was dialysed overnight against PBS, pH 7.3.The amount of antibody was quantified by measuring absorbance at 280 nmand the antibody concentration was calculated assuming that E₂₈₀^(0.1%)=1.4. Absorbance spectra analysis and SDS-PAGE were conducted onantibody fractions to verify their purity.

Example 3 Testing of Humanized Antibodies

3.1. Growth of Cells for Binding Analysis

HL-60 cells were obtained from the American Type Culture Collection(ATCC# CCL-240). They were maintained in a 5% CO₂ atmosphere, 37° C.water-jacketed incubator (Napco Scientific Co., Tualitan, Oreg.). Thecells were grown in RPMI media supplemented with L-glutamine(BioWhittaker, Walkersville, Md.) containing 10% fetal bovine serum(Hyclone, Logan, Utah), plus 50 μg/mL of gentamicin sulfate (LifeTechnologies, Grand Island, N.Y.).

3.2. Direct Binding Assay on HL-60 Cells

HL-60 cells were harvested from T75 flasks (NUNCLON™, cat. no. 153732),and centrifuged at about 300×g for five minutes in an Omnifuge RTtabletop centrifuge (Haereus Separations, Germany), at 4° C. The cellswere resuspended at a density of 3×10⁶ cells per mL in FACS buffer,comprised of 2% (v/v) goat serum (Sigma Chemical Co., St Louis, Mo.) inMEM Alpha Medium, supplemented with L-glutamine (Life Technologies,Grand Island, N.Y.). Using a multichannel pipetor, 3×10⁵ cells wereadded to wells of a Falcon 96-well round bottom plate (cat. no. 3077)and then placed on ice. Using a Falcon 96-well flexible assay plate(cat. no. 353911), eleven twofold serial dilutions of each test orcontrol article were prepared in duplicate in FACS buffer, from astarting concentration of 2×10⁻⁸ M. Then, 100 μl of either test orcontrol article was mixed with the cells. Control wells only receivedFACS buffer. The plate was incubated on ice for one hour, and thencentrifuged for five minutes at 300×g, in a refrigerated centrifuge.Reagents were removed from the plate wells by quick inversion over awaste beaker containing 10% bleach. The cells were resuspended by gentlevortexing, washed once with cold FACS buffer, centrifuged, and thenresuspended with gentle vortexing. FITC labeled goat-anti-human IgG(Jackson ImmunoResearch Laboratories, West Grove, Pa., cat. no.109-096-003) and FITC labeled goat-anti-mouse IgG (JacksonImmunoResearch Laboratories, West Grove, Pa., cat. no. 109-095-088) werediluted 1:100 in FACS buffer, and 100 μl was added to appropriate wellson the assay plate. The plate was then incubated for an additional houron ice, protected from light. Unbound secondary antibody was washed fromthe wells in the same manner as described above. The cells wereresuspended by gentle vortexing, and then fixed by adding 120 μl/well of1% formaldehyde in phosphate-buffered saline (PBS, 10 mM potassiumphosphate, 150 mM sodium chloride, pH 7.2). The assay plate was thenanalyzed on a FACScan flow cytometer interfaced with an automated96-well sampling device (Becton Dickinson, Mountain View, Calif.).

3.3. Preparation of Plasma Membranes

Plasma membranes were prepared according to the procedure described inVater et al. (1995). Briefly, HL-60 cells were grown to a density of1×10⁶ cells/mL in RPMI supplemented with L-glutamine, containing 10%fetal bovine serum, and 50 μg/mL of gentamicin sulfate. When a total of1×10⁹ cells was obtained, cells were harvested, spun at about 300×g inan Omnifuge RT centrifuge, washed with Hanks balanced salt solution(Life Technologies, cat. no. 14175-095), and combined into one 50 mLconical centrifuge tube. The pellet was then frozen at −80° C. for atleast 24 hours, to facilitate cell lysis and homogenization. To prepareplasma membranes, the pellet was thawed quickly at 37° C., and thenstored on ice; all subsequent steps were performed at 4° C. The HL-60cell pellet was resuspended in 8 mL of 10 mM Tris-HCl buffer, pH 7.0,containing 1 mM EDTA, 0.25 M sucrose, and 1 mMphenylmethylsulfonyl-fluoride (PMSF). The cell pellet was thentransferred to a 15 mL Dounce tissue grinder (Wheaton, Millville, N.J.)for cellular disruption (20 strokes with a tight fitting borosilicateglass pestle). The homogenate was centrifuged in a Sorval RC-5BRefrigerated Superspeed Centrifuge, using an SS-34 rotor (Wilmington,Del.) at 6000 rpm for ten minutes, and the supernatant carefully removedfrom the pellet. The pellet was washed an additional time with 8 mL ofbuffer and processed as described above. The combined supernatants werelayered over a 1 mL 37% sucrose cushion buffer in 10 mM Tris-HCl, 1 mMPMSF, 1 mM EDTA, pH 7.0, and processed further in a pre-chilled BeckmanL8-M ultracentrifuge, with an SW55ti rotor (Beckman Instruments, PaloAlto, Calif.), at 33,000 rpm for 70 minutes. The opalescent membranelayer, sitting just above the sucrose cushion, was removed from eachtube and diluted with four volumes of 10 mM Tris-HCl, 1 mM EDTA, 1 mMPMSF, pH 7.0. This solution was then centrifuged for an additional 30minutes at 18,000 rpm in the ultracentrifuge. The supernatant wascarefully drawn off, and the resultant pellets were resuspended in 10 mMTris-HCl, 1 mM EDTA, 1 mM PMSF, pH 7.0. The HL-60 membrane preparationwas aliquoted into 1.5 mL sterile screw cap Eppendorf tubes, frozen inliquid nitrogen, and then stored at −80° C. for use. The total proteinconcentration of the final preparation was determined using the Pierce™BCA Assay (Pierce Chemical Company, Rockford, Ill., cat. no. 23235).

3.4. Direct Binding Assay on HL-60 Membranes

HL-60 membranes, prepared as described above, were dried at a startingconcentration of 10 μg/mL in deionized water onto the polystyrenesurface of Immulon 2 96-well assay plates (Dynex Laboratories,Chantilly, Va.), using a Labconco vacuum desiccator (Labconco, Corp,Kansas City, Mo.), overnight at ambient temperature. The ELISA plate wasthen blocked with a 1% fraction V bovine serum albumin (Sigma-Aldrich,Inc., St. Louis, Mo., cat. no. A-3294), 0.05% Tween-20 (Sigma-Aldrich,Inc., St. Louis, Mo., cat. no. P-2287) solution in Tris-buffered saline(50 mM Tris-HCl, 150 mM sodium chloride, pH 7.5, TBS), 300 μL per well,at 37° C. for 1 hour. Following this blocking step, the plate wasdrained of blocking buffer and blotted onto paper towels. Two, threefoldserial dilutions of test or control reagents were prepared in blockingbuffer, in quadruplicate, starting at 3.13×10⁻⁹ M titrated down to5.29×10⁻¹⁴ M on a flexible 96-well assay plate. The negative controlwells contained blocking buffer alone. 50 μL of each dilution wastransferred to designated wells on the membrane-coated assay plate thatwas then incubated overnight at 4° C. The well contents were thenaspirated into a waste flask containing 10% (v/v) bleach, and the platewashed 3× with TBS containing 0.1% (v/v) Tween-20 (wash buffer) andblotted on paper towels. The amount of bound anti-My9-6 antibody wasdetected with either goat anti-mouse IgG-HRP or donkey anti-humanIgG-HRP diluted 1:1000 with blocking buffer in the appropriate wells.These secondary antibodies were incubated on the assay plate for onehour at room temperature, protected from light. The plate was washed andblotted as before. The plate was developed using TMB (BioFXLaboratories, Randallstown, Md., cat. no. TMBW-0100-01), then quenchedwith Stop solution (BioFX Laboratories, Randallstown, Md., cat. no.STPR-0100-01). The assay plate was read at A450 nm using a TITERTEK®Multiskan Plus MK II plate reader (Huntsville, Ala.).

3.5. Competition Binding Assay on HL-60 Membranes

HL-60 membranes, prepared as described above, were dried down from astarting concentration of 10 μg/mL in deionized water on the polystyrenesurface of Immulon 2 96-well assay plates (Dynex Laboratories,Chantilly, Va.), using a Labconco vacuum desiccator (Labconco, Corp,Kansas City, Mo.), overnight at room temperature. The ELISA plate wasthen blocked with a 1% fraction V bovine serum albumin (Sigma-Aldrich,Inc., St. Louis, Mo., cat. no. A-3294), 0.05% Tween-20 (Sigma-Aldrich,Inc., St. Louis, Mo., cat. no. P-2287) solution in Tris-buffered saline(50 mM tris, 150 mM sodium chloride, pH 7.5, TBS), 300 μl per well, at37° C. for 1 hour. Following this blocking step, the plate was drainedof blocking buffer and blotted onto paper towels. Two twofold serialdilutions of test or control reagents were prepared in blocking buffer,in quadruplicate, starting at 1.25×10⁻⁸ M titrated down to 2.44×10⁻¹¹ M(2× the final concentration needed) on a flexible 96-well assay plate.These unlabeled competing reagents were then mixed with an equal volumeof 2.5×10⁻¹⁰ M biotinylated murine anti-My9-6 (ImmunoGen, Inc.,Cambridge, Mass.); the positive control contained no competing reagent,whereas the negative control contained blocking buffer alone. 50 μL ofthese mixtures was transferred to designated wells on themembrane-coated assay plate that was then incubated overnight at 4° C.The well contents were then aspirated into a waste flask containing 10%(v/v) bleach, and the plate washed 3× with TBS containing 0.1% (v/v)Tween-20 (wash buffer). The plate was blotted onto paper towels, and theamount of bound biotinylated murine anti-My9-6 was detected with 100 μLper well of streptavidin-alkaline phosphatase (Jackson ImmunoResearchLaboratories, West Grove, Pa., cat. no. 016-050-084), diluted 1:5,000 inblocking buffer. Following a one hour incubation at room temperature andprotected from light, the unbound secondary antibody reagent was washedfrom the wells, and the plate was developed using TMB (BioFXLaboratories, Randallstown, Md., cat. no. TMBW-0100-01) quenched withStop solution (BioFX Laboratories, Randallstown, Md., cat. no.STPR-0100-01). The assay plate was read at A450 nm using a TITERTEK®Multiskan Plus MK II plate reader (Huntsville, Ala.).

3.6. Cloning Murine My9-6 Antibody Variable Regions

The murine My9-6 antibody variable regions were cloned by the RT-PCRmethods described above. Several individual light and heavy chain cloneswere sequenced to identify and avoid possible polymerase generatedsequence errors. Only a single sequence was obtained for the lightchain, but two separate sequences were pulled out of the heavy chainRT-PCR clones.

To confirm the actual sequences of the murine My9-6 light chain variableregion, peptide sequences were analyzed by Edman degradation, MatrixAssisted Laser Desorption Ionisation (MALDI) and Tandem MassSpectrometry (MS-MS). The 22 residues at the amino terminus of the My9-6light chain were sequenced by Edman degradation and are given in FIG.6A. The initial cDNA sequence derived from degenerate RT-PCR clonesmatched all but four residues that were likely generated by thedegenerate primers. The peptide sequence determined by Edman degradationwas later confirmed by RT-PCR clones generated by degenerate primers inthe 5′ end signal peptide sequence. MS-MS analysis of tryptic digestfragments also confirmed the sequence of the light chain CDRs 1 and 2(FIG. 6B). Together, these peptide analyses confirm the predicted murineMy9-6 light chain amino acid sequence derived from the RT-PCR generatedcDNA clones.

As is common with heavy chain sequences, the muMy9-6 heavy chain aminoterminus was blocked by a pyroglutamine residue and therefore could notbe sequenced by N-terminal Edman degradation. Instead internal sequencedata was generated by tryptic digests and analysis by Matrix AssistedLaser Desorption Ionisation (MALDI) and Tandem Mass Spectrometry(MS-MS). A 1788 dalton fragment was identified with a predicted sequenceidentical to a portion of cDNA clone 2 (FIG. 7). This sequence includesfive residues from CDR3 for cDNA clone 2, and therefore is an idealpeptide to make a positive identification for muMy9-6 heavy chain.Multiple RACE PCR clones producing only the sequence originally obtainedfrom the degenerate RT-PCR clone 2 also confirmed this peptide sequence.

The cumulative results from the various cDNA clones and the peptidesequence analysis provided the final murine My9-6 light and heavy chainsequences presented in FIG. 8. Using Kabat and AbM definitions, thethree light chain and heavy chain CDRs were identified (FIGS. 8 and 9).A search of the NCBI IgBlast database indicates that the muMy9-6antibody light chain variable region most likely derives from the mouseIgVκ8-27 germline gene while the heavy chain variable region most likelyderives from the IgVh V102 germline gene (FIG. 10).

3.7 Determination of the Variable Region Surface Residues of My9-6Antibody

The antibody resurfacing techniques described by Pedersen et al. (1994,J. Mol. Biol. 235:959-973) and Roguska et al. (1996, Protein Eng.9:895-904) begin by predicting the surface residues of the murineantibody variable sequences. A surface residue is defined as an aminoacid that has at least 30% of its total surface area accessible to awater molecule. In the absence of a solved structure to find the surfaceresidues for muMy9-6, the ten antibodies with the most homologoussequences in the set of 127 antibody structure files were aligned (FIGS.11A & B). The solvent accessibility for each Kabat position was averagedfor these aligned sequences and the distribution of the relativeaccessibilities for each residue are presented in FIG. 12.

Several surface positions had average accessibilities of between 25% and35%. These were looked at more closely by averaging only the antibodieswith two identical residues flanking on either side (FIGS. 13A and B).The 25 predicted surface residues for the muMy9-6 heavy chain wereunchanged after the additional analysis, but Kabat positions 15 and 70in the light chain required further consideration. The alanine at lightchain position 15 had an average accessibility of 34.4% in all tenstructures, but only a single structure, 1mcp, had identical flankingresidues to those in muMy9-6. In this structure, Ala15 had a relativeaccessibility of only 18.2%. Because in this single structure Ala15 wasdramatically less accessible than the average residue for position 15,the three structures with only a single difference in the flankingresidues (1ap2, 1frg, and 1hil) were also averaged together. The averageaccessibility for these three structures was again 33.3%, making Ala15 apredicted surface residue. Finally, resurfaced anti-B4, C242, and theresurfacing patent itself predict that light chain position 15 is asurface residue. Taken together, the conservative approach is to assumethat muMy9-6 light chain position 15 is a surface residue.

The muMy9-6 light chain position 70 also required special considerationsthat ultimately resulted in a conservative prediction of it being asurface residue. The initial surface prediction work for the light chainwas done with MC data from only the five most homologous light chainstructures, and in this group Asp70 was a predicted surface residue.Later, with the additional five most homologous structures, position 70was found to have an average accessibility of 29.1% as shown in FIG.13A. In addition, nine of the ten structures had the identical flankingresidues and the average of this set was 29.2% accessible. Because thisresidue appeared to be so close to the 30% cut off and the resurfacingpatent calls position 70 a surface position, the accessibility data wasfurther scrutinized. Position 70 for the structure 1 lve has a relativeaccessibility of 22.5%, which is five percentage points less than thenext lowest relative accessibility of 27.2%. Since 1lve is the eighthmost homologous structure and it was a low outlier, an average was takenfor the eight structures with identical flanking residues besides 1lveand this set had a 30.1% average accessibility. This, together with thefact that the initial muMy9-6 light chain analysis and the resurfacingpatent call light chain position 70 a surface position, led to theconservative prediction that the muMy9-6 light chain Asp70 is a surfaceresidue.

3.8. Molecular Modeling to Determine Which Residues Fall Within 5 Å of aCDR

The molecular model, generated with the AbM software package, wasanalyzed to determine which muMy9-6 surface residues come within 5 Å ofa CDR. In order to resurface the antibody, all surface residues outsideof a CDR must be changed to it's human counterpart, but residues thatcome within 5 Å of a CDR may also contribute to antigen binding andspecificity. Therefore these 5 Å residues must be identified andconsidered throughout the humanization process. The CDR definitions usedfor resurfacing combines the AbM definition for heavy chain CDR2 andKabat definitions for the remaining five CDRs (FIG. 9). The murine modelwas analyzed with the Swiss Viewer Software and FIG. 14 shows theresidues that are within 5 Å of any CDR residue in either the light orheavy chain sequence.

3.9. Selection of the Most Homologous Human Surface

Candidate human antibody surfaces for resurfacing muMy9-6 were pulledfrom the Kabat antibody sequence database using SR software. Thissoftware provides an interface to search only specified residuepositions against the antibody database. To preserve the natural pairs,the surface residues of both the light and heavy chains were comparedtogether. The most homologous human surfaces from the Kabat databasewere aligned in order of rank of sequence identity. The top fivesurfaces as aligned by the SR Kabat database software are given in FIG.15. The surfaces were then compared to identify which human surfaceswould require the least changes within 5 Å A of a CDR. Theanti-Hepatitis C Virus antibody, LC3bPB (Ivanovski M. et al., 1998,Blood 91(7):2433-42), requires the least number of surface residuechanges (10 total) and only four of these residues come within 5 Å of aCDR. The full length variable region sequence for muMy9-6 was alsoaligned against the Kabat human antibody database with SR and LC3bPB wasidentified as the 14th most similar human variable region sequence (datanot shown). Since the LC3bPB antibody provides the most homologoussurface and requires the least number of 5 Å residue changes, it is thebest candidate to resurface muMy9-6.

3.10. Construction of the My9-6 Genes for Humanized My9-6 Antibodies

The ten surface residue changes for huMy9-6 were made using PCRmutagenesis techniques as described above. Since six of the surfaceresidues for LC3bPB were more than 5 Å from a CDR, these residues werechanged from murine to human in all versions for humanized My9-6 (FIGS.16A and B). The four surface residues that did fall within 5 Å of a CDR,Kabat light chain positions 1, 3 and 45 and heavy chain position 64,were either changed to human or retained as murine to make up the 16humanized versions of My9-6. Of these, the most human is version 1.0since it has all ten human surface residues. The most conservativeversion in terms of ensuring maximum binding affinity is version 1.1which retains the four murine surface residues that are within 5 Å of aCDR. Each of the humanized My9-6 antibody genes were cloned into theantibody expression plasmid (FIG. 5) for transient and stabletransfections.

3.11. Binding Data

The key strength of humanization by resurfacing is that by retaining thenon-surface murine framework residues, humanized My9-6 should continueto bind CD33 with unchanged affinity. However, the four surface residuesthat come within 5 Å of a CDR may contribute to antigen binding and,therefore, changing these residues could have a negative effect onbinding. To address this concern, the 16 versions of humanized My9-6include all combinations of either the human or murine residues at eachof the four 5 Å residue positions. These range from the optimal or mosthuman version, 1.0, with human residues at every non-CDR surfaceposition, to the most conservative version, 1.1, which retains themurine residues in each of the four 5 Å surface positions and,therefore, should retain wild-type binding. By comparing the bindingaffinity of the humanized My9-6 versions to murine My9-6, the most humanversion that retains wild-type binding can be selected as theresurfaced, humanized My9-6.

Direct and competitive binding experiments with the humanized versionsof murine My9-6 and with muMy9-6 were done on the CD33 expressing humanleukemic cell line, HL-60. The three most human versions (V1.0, V1.3,V1.6) as well as the most conservative version (V1.1) were tested oneither HL-60 membranes or whole cells. FIG. 17 gives the KD values foreach condition and FIG. 18 shows example binding curves for huMy9-6,V1.0, versus muMy9-6. The KD values calculated for the humanized My9-6versions including the most human, version 1.0, fall within theexperimental error of the KD value of murine My9-6. The competitivebinding results also demonstrate that huMy9-6 antibodies can competeequally well for CD33 binding as muMy9-6 itself. With these datademonstrating near wild type binding affinities for the fully humanizedMy9-6, V1.0, on HL-60 membranes and whole cells, there was no need totest additional versions, since version 1.0 is the optimal humanizedMy9-6 antibody.

The murine My9-6 antibody has been fully humanized without loss ofbinding activity. None of the surface residue changes from mouse tohuman resulted in a loss of binding affinity, even though four of thesesurface residues were within 5 Å of a CDR. Residues within 5 Å of a CDRare thought to make potentially critical van der Waal contacts that mayaffect the structure of these CDRs.

The results of the My9-6 humanization suggest that as the numbers ofresurfaced antibodies grow, a thorough residue position analysis basedon actual binding data might become a more effective way of targetingquestionable residues than simply looking to any residue within 5 Å of aCDR.

Humanized My9-6 V1.0 (huMy9-6) is the fourth antibody that was humanizedusing the procedure described herein. Humanization by resurfacing of thevariable region framework of a murine antibody was developed as animprovement upon standard humanization methods, including CDR grafting,which can require extensive development and reengineering to maintainbinding activity. This is contrasted by the resurfaced huMy9-6 antibody,which involved changing only ten surface residues to create a fullyactive antibody with a completely human surface.

Example 4 My9-6-DM1 Immunoconjugate

Preclinical efficacy studies with human tumor xenografts in mice usingthe My9-6 antibody conjugated to the maytansinoid drug DM1 wereperformed.

As discussed below, My9-6-DM1 maintained the specificity and bindingaffinity of the unmodified antibody for CD33, and showed potentantigen-specific cytotoxicity toward CD33-positive tumor cells in vitro.Treatment of SCID mice bearing established subcutaneous HL-60 tumorxenografts with My9-6-DM1, resulted in complete eradication of thetumors at doses (20 mg conjugated antibody/kg/day i.v.×5 days) whichshowed no toxicity and were well below the maximum tolerated dose. Incontrast, treatment with My9-6 antibody alone had no effect on HL-60tumor growth compared to vehicle control. Similar curative efficacy wasobserved with My9-6-DM1 upon treatment of mice bearing subcutaneousTHP-1 xenografts.

4.1. Reagents

The antibody was the murine monoclonal My9-6 antibody directed againsthuman CD33 described above.

The antibody modifying agent was N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP):

The cytotoxic drug was the maytansine analog, DM1, which is synthesizedfrom a microbial fermentation product, Ansamitocin P3. DM1 is a potentinhibitor of tubulin polymerization.

4.2. Preparation of My9-6-DM1

My9-6-DM1 was prepared by modifying the My9-6 antibody with SPP tointroduce 3-5 pyridyldithio groups per molecule of antibody. Disulfideexchange between the thiol substituent on the DM1 and the activedisulfide functionality on the antibody provided the DM1-containingantibody conjugate.

(n may be any integer)

4.3. Analysis of Antibody and Antibody Conjugate Binding byFlow-Cytometry

HL-60 cells were incubated with various concentrations of either My9-6antibody or My9-6-DM1. The cells were then washed and incubated with asecondary FITC-labeled anti-murine IgG antibody. Following an additionalwash, cells were fixed with 2% formaldehyde and cell-associatedfluorescence was quantified using a BD FACScan flow cytometer. My9-6-DM1maintains specific CD33 binding comparable to the unmodified antibody(FIG. 19).

4.4. In Vitro Cytotoxicity Assay

The cytotoxicity of My9-6-DM1 was measured using the CD33-expressingTHP-1 cell and the CD33-negative cell line Namalwa. Cells were plated in96-well plates in media containing the conjugate and incubated at 37° C.until colonies had formed (2-3 weeks). Colonies were then scored, andthe surviving fractions determined using Poisson distribution in thecalculations.

4.5. In Vivo Mouse Tumor Xenograft Studies

Subcutaneous tumor model—HL-60 human promyelocytic leukemia cells (5×10⁶cells in 0.1 mL) were subcutaneously inoculated into the right flank offemale SCID mice. Mice were treated by intravenous injection into alateral tail vein starting when tumors reached 100 mm³ in size (400 mm³in large tumor model). Tumor size and mouse body weight were measuredtwice per week. Survival model—HL-60 cells (5×10⁶ cells in 0.1 mL) wereintravenously injected into the lateral tail vein of female SCID mice.Eleven days after tumor cell injection treatment was started. Mice werechecked every day for moribund, tumor mass, or death. Body weight wasmeasured twice per week.

4.6. In Vitro Specificity and Efficacy of My9-6-DM1

The cytotoxicity of My9-6-DM1 toward CD33 expressing cells (THP-1)compared to a CD33-negative cell line (Namalwa) was tested using aclonogenic assay, where cell killing activity is determined byquantifying the number of colonies that can grow following treatment.My9-6-DM1 exhibits potent cell killing activity toward CD33-positivehuman tumor cells in vitro (FIG. 20). No significant toxicity towardCD33-negative cells was observed, indicating that the CD33-dependentcytotoxicity was due to specific targeting by the anti-CD33 antibody,My9-6.

4.7. Efficacy of My9-6-DM1 Against Human Tumor Xenografts in Mice

The efficacy of My9-6-DM1 in vivo was determined using SCID mice bearinghuman HL-60 tumor cell xenografts. HL-60 cells were injectedsubcutaneously and tumors were allowed to grow to an average size of 100mm³. My9-6-DM1 conjugate was delivered i.v. once a day for 5 days at thedoses indicated in FIG. 21. Dosage is expressed as mg antibody in theconjugate, which corresponds to a DM1 dose of approximately 15 μg DM1per mg of antibody. Tumor volume was measured as an indication oftreatment efficacy and mouse body weight was monitored to indicatetoxicity due to treatment. My9-6-DM1 induces long-term cures of micebearing human HL-60 cell xenografts at doses that cause little toxicity(FIG. 21). At the two highest doses tested (19 and 26 mg/kg), completeregression of the tumors was observed with a maximum body weight loss ofless than 10% (FIGS. 21A and B). Even at the lowest dose tested (13mg/kg), 2 of 5 mice showed complete cures, while the other 3 mice showedtumor regression with tumor growth delayed by approximately 20 days.Similar results were also observed using the THP-1 leukemic cell linewhere My9-6-DM1 induced long-term cures of mice bearing human THP-1tumor xenografts (data not shown).

These results are in sharp contrast to those obtained by treatment withMy9-6 antibody alone. Antibody alone has no effect on tumor cell growtheven at 50 mg/kg (FIG. 21C).

The activity of My9-6-DM1 was compared with the approved anti-CD33antibody-calicheamicin conjugate Gemtuzumab ozogamicin. Gemtuzumabozogamicin was administered every 4 days for 3 doses. A preliminaryexperiment demonstrated that Gemtuzumab ozogamicin at the published MTDfor nude mice (0.3 mg/kg) was too toxic for SCID mice (data not shown).Significant toxicity was also observed at the 100 and 200 μg/kg dose inthis experiment (FIG. 21F). Gemtuzumab ozogamicin showed only modesttumor regression and growth delay in this model (about 25 days) at themaximum tolerated dose (100 μg/kg) (FIG. 21E).

In a separate experiment, the anti-tumor activity of the My9-6-DM1conjugate was compared to the activity of the unconjugated parent drug,maytansine. Mice bearing HL-60 tumor xenografts were treated daily for 5days with conjugate at 23 mg antibody/kg or 350 μg DM1/kg; unconjugateddrug at 350 μg/kg; or unconjugated antibody at 23 mg/kg. Three of 6 micetreated with maytansine alone died two days after treatment indicatingthat this level of drug is quite toxic. However, the drug showed asignificant impact on tumor growth in the remaining 3 mice, with a delayin growth of approximately 10 days (FIG. 22).

My9-6 antibody alone had no affect on tumor growth, while My9-6-DM 1conjugate caused complete regression of the tumors. The effect ofconjugate treatment on mouse body weight (12% decrease) was slightlygreater than in the previous experiment and may reflect batch to batchvariation in conjugate synthesis. Regrowth of tumors in two of the sixconjugate-treated mice was observed starting at day 70. These animalswere subjected to a second treatment of My9-6-DM1 identical to the firsttreatment. The second treatment resulted in the complete regression ofthe “relapsing” tumors with no adverse toxicity observed, suggestingthat the regrowth of tumor was not due to the growth ofconjugate-resistant cells.

The potent efficacy of My9-6-DM1 in this xenograft model suggested thatthe conjugate may be effective even against large HL-60 tumors. Tumorswere allowed to grow to a tumor volume of >400 mm³ prior to theinitiation of treatment. In addition, a comparison of the activity ofMy9-6-DM1 with standard chemotherapeutic drugs was conducted. My9-6-DM1caused complete regression of large tumors in SCID mice (FIGS. 23A & B).Again, relapsed tumors (2 of 6 mice) were sensitive to a secondtreatment of My9-6-DM1. Treatment with standard chemotherapeutic agents(Ara-C and idarubicin) had little effect on tumor growth. Higher drugdoses were profoundly toxic to the mice (not shown).

4.8. Efficacy of My9-6-DM1 in HL-60 Cell Mouse Survival Model

While the efficacy of My9-6-DM1 in the tumor xenograft model isstriking, it is also of interest to look at the activity of theconjugate in a mouse survival model where HL-60 cells are injecteddirectly into a mouse tail vein. In this model, control mice die between30 and 50 days after cell injection. Mice treated with My9-6-DM1starting 11 days after cell injection showed a dramatic increase insurvival, with 7 of 8 mice alive at 70 days (FIG. 24A). Maytansine alonealso showed significant impact on mouse survival in this model, though 2of 8 mice died shortly after the start of treatment indicating drugtoxicity. The relative efficacy of maytansine in this model compared tothe subcutaneous xenograft model suggests that HL-60 tumors may be moresensitive to the action of the drug in this setting. A further delay inthe start of treatment may demonstrate a sharper differential betweentargeted and untargeted drug. However, even in this model, standardchemotherapy provided no significant survival enhancement (FIG. 24B).The highest combination dose showed significant apparent drug toxicity.Both My9-6 antibody alone and Gemtuzumab ozogamicin showed a modestincrease in survival.

Statement of Deposit

The hybridoma that makes murine My9-6 antibodies was deposited with theAmerican Type Culture Collection, PO Box 1549, Manassas, Va. 20108, onNov. 7, 2002, under the Terms of the Budapest Treaty and was assigneddeposit number PTA-4786.

Certain patents and printed publications have been referred to in thepresent disclosure, the teachings of which are hereby each incorporatedin their respective entireties by reference.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

1. An isolated antibody or fragment thereof, comprising at least onecomplementarity-determining region having an amino acid sequenceselected from the group consisting of SEQ ID NOs:1-6.
 2. An antibody orfragment thereof, comprising at least one heavy chain variable regionand at least one light chain variable region, wherein said heavy chainvariable region comprises at least one complementarity-determiningregion having the amino acid sequence of SEQ ID NOs:1, 2 or 3, andwherein said light chain variable region comprises at least onecomplementarity-determining region having the amino acid sequencesrepresented by SEQ ID NOs:4, 5 or
 6. 3. The antibody or fragment thereofof claim 2, wherein said heavy chain variable region has at least 90%sequence identity to an amino acid sequence represented by SEQ ID NO:7.4. The antibody or fragment thereof of claim 2, wherein said heavy chainvariable region has at least 95% sequence identity to said amino acidsequence represented by SEQ ID NO:7.
 5. (canceled)
 6. The antibody orfragment thereof of claim 2, wherein said light chain variable regionhas at least 90% sequence identity to an amino acid sequence representedby SEQ ID NO:8.
 7. The antibody or fragment thereof of claim 2, whereinsaid light chain variable region has at least 95% sequence identity tosaid amino acid sequence represented by SEQ ID NO:8.
 8. (canceled) 9.The antibody or fragment thereof of claim 2, wherein said heavy chainvariable region has at least 90% sequence identity to an amino acidsequence represented by SEQ ID NO:9.
 10. The antibody or fragmentthereof of claim 2, wherein said heavy chain variable region has atleast 95% sequence identity to said amino acid sequence represented bySEQ ID NO:9.
 11. (canceled)
 12. The antibody or fragment thereof ofclaim 2, wherein said light chain variable region has at least 90%sequence identity to an amino acid sequence represented by SEQ ID NO:10.13. The antibody or fragment thereof of claim 2, wherein said lightchain variable region has at least 95% sequence identity to said aminoacid sequence represented by SEQ ID NO:10. 14-16. (canceled)
 17. Animmunoconjugate comprising the antibody or epitope-binding fragmentthereof of claim 1 linked to a drug or prodrug.
 18. An immunoconjugatecomprising the antibody or epitope-binding fragment thereof of claim 2linked to a drug or prodrug.
 19. The immunoconjugate of claim 17,wherein said drug or prodrug is selected from the group consisting of amaytansinoid, a taxoid, CC-1065, a CC-1065 analog, dolastatin, adolastatin analog, methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, andderivatives thereof.
 20. The immunoconjugate of claim 18, wherein saiddrug or prodrug is selected from the group consisting of a maytansinoid,a taxoid, CC-1065, a CC-1065 analog, dolastatin, a dolastatin analog,methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine,melphalan, mitomycin C, chlorambucil, calicheamicin, and derivativesthereof.
 21. A composition comprising the antibody or epitope-bindingfragment thereof of claim 1 and a drug or prodrug.
 22. A compositioncomprising the antibody or epitope-binding fragment thereof of claim 2and a drug or prodrug.
 23. A pharmaceutical composition comprising theantibody or epitope-binding fragment thereof of claim 1, and apharmaceutically acceptable agent.
 24. A pharmaceutical compositioncomprising the antibody or epitope-binding fragment thereof of claim 2,and a pharmaceutically acceptable agent.
 25. A pharmaceuticalcomposition comprising the immunoconjugate of claim 17, and apharmaceutically acceptable agent.
 26. A pharmaceutical compositioncomprising the immunoconjugate of claim 18, and a pharmaceuticallyacceptable agent.
 27. A pharmaceutical composition comprising thecomposition of claim 21, and a pharmaceutically acceptable agent.
 28. Apharmaceutical composition comprising the composition of claim 22, and apharmaceutically acceptable agent.
 29. A diagnostic reagent comprisingthe antibody of claim 1, wherein said antibody or antibody fragment islabeled.
 30. A diagnostic reagent comprising the antibody of claim 2,wherein said antibody or antibody fragment is labeled.
 31. Thediagnostic reagent of claim 29, wherein said label is selected from thegroup consisting of a biotin label, an enzyme label, a radio-label, afluorophore, a chromophore, an imaging agent and a metal ion.
 32. Thediagnostic reagent of claim 30, wherein said label is selected from thegroup consisting of a biotin label, an enzyme label, a radio-label, afluorophore, a chromophore, an imaging agent and a metal ion.
 33. Amethod for inhibiting the growth of a cell expressing CD33 comprisingcontacting said cell with the antibody or epitope-binding fragmentthereof of claim 1 or
 2. 34. A method for inhibiting the growth of acell expressing CD33 comprising contacting said cell with theimmunoconjugate of claim 17 or
 18. 35. A method for inhibiting thegrowth of a cell expressing CD33 comprising contacting said cell withthe composition of claim 21 or
 22. 36. A method for inhibiting thegrowth of a cell expressing CD33 comprising contacting said cell with apharmaceutical composition selected from claims 23-28.
 37. A method fortreating a subject having a disease wherein CD33 is expressed,comprising administering to said subject an effective amount of theantibody or epitope-binding fragment thereof of claim 1 or
 2. 38. Amethod for treating a subject having a disease wherein CD33 isexpressed, comprising administering to said subject an effective amountof the immunoconjugate of claim 17 or
 18. 39. A method for treating asubject having a disease wherein CD33 is expressed, comprisingadministering to said subject an effective amount of the composition ofclaim 21 or
 22. 40. A method for treating a subject having a diseasewherein CD33 is expressed, comprising administering to said subject aneffective amount of the pharmaceutical composition of claim 23 or 24.41. A method for treating a subject having a disease wherein CD33 isexpressed, comprising administering to said subject an effective amountof the pharmaceutical composition of claim 25 or
 26. 42. A method fortreating a subject having a disease wherein CD33 is expressed,comprising administering to said subject an effective amount of thepharmaceutical composition of claim 27 or
 28. 43. A method for treatinga subject having a disease wherein CD33 is expressed, comprisingcontacting one or more cells of said subject ex vivo with an effectiveamount of the antibody or epitope-binding fragment thereof of claim 1 or2.
 44. A method for treating a subject having a disease wherein CD33 isexpressed, comprising contacting one or more cells of said subject exvivo with an effective amount of an immunoconjugate of claim 17 or 18.45. A method for treating a subject having a disease wherein CD33 isexpressed, comprising contacting one or more cells of said subject exvivo with an effective amount of a composition of claim 21 or
 22. 46. Amethod for treating a subject having a disease wherein CD33 isexpressed, comprising contacting one or more cells of said subject exvivo with an effective amount of a pharmaceutical composition selectedfrom claims 23-28.
 47. The method of treatment of claim 37, wherein saiddisease is a disease selected from the group consisting ofmyelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML).
 48. The methodof treatment of claim 38, wherein said disease is a disease selectedfrom the group consisting of myelodysplastic syndrome (MDS), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).
 49. The method of treatment of claim 39,wherein said disease is a disease selected from the group consisting ofmyelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML).
 50. The methodof treatment of claim 40, wherein said disease is a disease selectedfrom the group consisting of myelodysplastic syndrome (MDS), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).
 51. The method of treatment of claim 41,wherein said disease is a disease selected from the group consisting ofmyelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML).
 52. The methodof treatment of claim 42, wherein said disease is a disease selectedfrom the group consisting of myelodysplastic syndrome (MDS), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).
 53. The method of treatment of claim 43,wherein said disease is a disease selected from the group consisting ofmyelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML).
 54. The methodof treatment of claim 44, wherein said disease is a disease selectedfrom the group consisting of myelodysplastic syndrome (MDS), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).
 55. The method of treatment of claim 45,wherein said disease is a disease selected from the group consisting ofmyelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML) and pro-myelocytic leukemia (PML).
 56. The methodof treatment of claim 46, wherein said disease is a disease selectedfrom the group consisting of myelodysplastic syndrome (MDS), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML) andpro-myelocytic leukemia (PML).
 57. A method of determining whether abiological sample contains a myelogenous cancer cell, comprising: (a)contacting said biological sample with a diagnostic reagent of claim 29or 30, and (b) detecting the distribution of said reagent within saidsample.
 58. The method of diagnosis of claim 57, wherein said cancer isa cancer selected from the group consisting of acute myeloid leukemia(AML), chronic myeloid leukemia (CML) and pro-myelocytic leukemia (PML).59. An improved antibody or fragment thereof that specifically binds toCD33, said improved antibody or antibody fragment prepared by: (a)providing a DNA that encodes an antibody or epitope-binding fragmentthereof comprising at least one of SEQ ID NO:7 and SEQ ID NO:8, (b)introducing at least one nucleotide mutation, deletion, insertion oraddition into said DNA such that the amino acid sequence of saidantibody or epitope-binding fragment encoded by said DNA is changed; (c)expressing said antibody or epitope-binding fragment; (d) screening saidexpressed antibody or epitope-binding fragment for said improvement,thereby preparing an improved antibody or epitope-binding fragment. 60.An improved antibody or epitope-binding fragment thereof thatspecifically binds to CD33, said improved antibody or antibody fragmentprepared by: (a) providing a DNA that encodes an antibody orepitope-binding fragment thereof comprising at least one of SEQ ID NO:9and SEQ ID NO:10, (b) introducing at least one nucleotide mutation,deletion, insertion or addition into said DNA such that the amino acidsequence of said antibody or epitope-binding fragment encoded by saidDNA is changed; (c) expressing said antibody or epitope-bindingfragment; (d) screening said expressed antibody or epitope-bindingfragment for said improvement, thereby preparing an improved antibody orepitope-binding fragment.
 61. The improved antibody or antibody fragmentof claim 59 or 60, wherein said improvement is an increased affinity forCD33.
 62. The improved antibody or antibody fragment of claim 59 or 60,wherein said at least one nucleotide mutation, deletion, insertion oraddition is made by a method selected from the group consisting ofoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling and use of mutator-strainsof E. coli.
 63. An isolated polynucleotide encoding the antibody orepitope-binding fragment thereof of claim 1 or
 2. 64. An isolatedpolynucleotide encoding a light or heavy chain of the antibody orepitope-binding fragment thereof of claim 1 or
 2. 65. A recombinantvector comprising the polynucleotide of claim
 63. 66. A recombinantvector comprising the polynucleotide of claim
 64. 67. A host celltransformed with the recombinant vector of claim
 65. 68. A host celltransformed with the recombinant vector of claim
 66. 69. A method forproducing an antibody or epitope-binding fragment thereof having theability to bind CD33, said method comprising (a) culturing a host cellas claimed in claim 67 under conditions such that said host cellexpresses the antibody or epitope-binding fragment, and (b) collectingthe antibody or epitope-binding fragment so expressed.
 70. A method forproducing an antibody or epitope-binding fragment thereof having theability to bind CD33, said method comprising (a) culturing a host cellas claimed in claim 68 under conditions such that said host cellexpresses the antibody or epitope-binding fragment, and (b) collectingthe antibody or epitope-binding fragment so expressed.
 71. A method forobtaining CD33 from a biological material, said method comprising: (a)contacting a biological material with the antibody or epitope-bindingfragment thereof of claim 1 or 2, (b) permitting the antibody orepitope-binding fragment of claim 1 or 2 to bind to CD33 in saidbiological material, and (c) isolating the antibody or epitope-bindingfragment bound to CD33 from the biological material, thereby obtainingCD33 from a biological material.